Extremophiles are valuable resources in biotechnology. These organisms are adapted to grow at 100° C. in hot springs, at low temperature in cold polar seas, at high pressure in deep sea, at a very low and high pH values (pH 0-1 or pH 10-14) and at very high salt concentration up to 35%. Extremophiles and their cell components have been used in various industries as well as in environmental biotechnology. Industrial applications of alkaliphiles include exploitation of enzymes such as proteases, amylases, cellulases, and lipases which are commercialized and widely used mainly in detergent industries and bioremediation. Also, enzymes from extremophiles are expected to fill the gaps between biological and chemical processes due to their unusual properties.
Alkaline environment is a typical extreme environment which includes naturally occurring soda lakes, deserts, carbonated soil and industrial waste. The physiological properties of alkaliphilic microorganisms offer a multitude of potential applications in various fields of biotechnology and their utilization for commercial purposes. To employ the right bacterial culture for its exploitation, it is necessary to isolate them in pure form, which is only possible on solid media. To prepare solid media for alkaliphiles, it is necessary to add a gelling agent, which is stable in extreme alkaline condition. Apart from this, to study the biodiversity of cultivable alkaliphiles, it is necessary to purify them from mixed culture as individual colonies for which they are required to grow on a solid medium so that they grow as individual clones.
The use of solid culture media has been of fundamental importance to microbiological research since the nineteenth century. Desirable qualities of a solidifying agent for media include stability of gel over the temperature range of bacterial growth, resistance to digestion by bacteria, lack of syneresis, transparency, and the ability to form a reversible colloid. The solid culture medium must be firm enough to facilitate streaking and spreading. In addition, it is desirable that the gelling agent is relatively inexpensive and easily available. Since Koch first introduced agar as a gellifying agent in bacteriological media (Koch, R. Die Atiologie der Turberkulose. Berl. Klin. Wochenschr. (1882) 19: 221-230), it has become the primary gelling agent for solid media throughout the world. However, increased cost and shortage of resources have made a more readily available substitute for agar, desirable.
Carrageenan is a family of linear sulphated polysaccharides extracted from red seaweeds. Three basic types of carrageenan are available, which differ in the numbers and location of sulfated ester. These chemicals are large, highly flexible molecules, which curl around each other forming double helical structures in the presence of monovalent and divalent cations. This gives them the ability to form a variety of thermo-reversible gels at room temperature. Large scale cultivation of carrageenophytes is successfully carried out all over the world including India and hence there is no shortage of carrageenan yielding seaweeds. Good cultivation practices and simple extraction procedures leads carrageenan, a cheaper gelling agent as compared to agar. Carrageenan is widely used in the food and pharmaceutical industries as thickening, stabilizing and gelling agents.
A. W. Walker and A. A. Day in their paper entitled “Extracts from Irish moss as a substitute for bacteriological culture media” in Food Res. (1943) 8: 435-443 have described the extraction of carrageenan from Irish moss. They prepared the bacteriological media using agar and extracts of Irish moss as gelling agents and autoclaved and dispensed to plates. They observed the similar bacterial growth after 24 h incubation on the plates and also noted lack of syneresis with low concentration of carrageenan. However, they have introduced carrageenan as a substitute of agar for bacteriological culture media. The drawback is no mention made on pH of the media. They did not describe the media preparation having alkaline pH and isolation of alkaliphiles.
N. Watson and D. Apirion in their paper entitled “Substitute for agar in solid media for common usage in microbiology” in Appl. Env. Microbiol. (1976) 31: 509-513 have prepared media using various concentrations (1 to 4%) of commercially available gelling agents like agar, gelatin types I and II, Ca++ and K+ salts of carrageenan and autoclaved and allowed to solidify in small depression plates and incubated at 25 and 45° C. for gelling study. According to them, all the concentrations of gelatin types gave soft gel, where as all the concentrations of the Ca2+ salt of carrageenan gave firm gel at 25° C., but soft gel at 45° C. while K2+ salt of carrageenan gave firm gel at 25 and 45° C. They prepared enriched media containing either 1.5% agar or 2% carrageenan for growth comparison study of mutant E. coli stains. They observed that the patched colonies grew similarly on both the plates and reported the plates containing carrageenan were clearer and the colonies were more distinguishable. The drawbacks of the paper are the cultivation of mutant E. coli strains on carrageenan containing media and they have not mentioned about pH of the bacteriological media. They did not describe the preparation of solid carrageenan media in extreme alkaline pH and no mention is made on the cultivation of alkaliphilic bacteria.
A. D. Lines in his paper entitled “Value of the K+ salt of carrageenan as an agar substitute in routine bacteriological media” in Appl. Env. Microbiol. (1977) 34: 637-639 has described the preparation of media having pH 7.6 with two different gelling agents, i.e. agar (1.2 to 2%) and carrageenan (2%). He assessed the suitability of carrageenan containing media by studying the parameters like (i) appearance, (ii) gel strength, (iii) ease of preparation, (iv) growth support including retention of antigenic characteristics, (v) resistance to digestion, and (vi) utilization of components by microorganisms. He reported 2% suspension of carrageenan retained its gel strength at 45° C. than their respective agar. However, 2% carrageenan looses its gel strength at 60° C. where higher concentration (2.4% carrageenan) overcomes the problem. He also mentioned that melting and gelling temperatures of carrageenan is similar to those of agar. He concluded that carrageenan containing media supports the growth of microbes with colony morphology, time of growth, viability, and pigment productions which were identical to those of agar preparations. The drawbacks are the preparation of bacteriological media using agar and carrageenan having pH 7.6 and comparison of only colony characteristics on both the media. However, he did not describe the preparation of alkaline solid media having pH greater than 7.6 and no mention is made on the cultivation of alkaliphilic bacteria.
Epifanio et al in their paper entitled “Carrageenan from Eucheuma striatum (Schmitz) in bacteriological media” in Appl. Env. Microbiol. (1981) 41: 155-158 have described the bacteriological media composed of either agar or carrageenan as gelling agent, 15 g; with peptone, 5 g; sodium chloride, 8 g; beef extract, 3 g; distilled water 1000 ml. The above media were autoclaved and used to prepare solid plates, slants and butts. Bacterial cultures were inoculated and incubated under conditions required by the organisms. They studied that the carrageenan and agar gels were sufficiently firm to permit effective and convenient streaking on plates and slants, requiring a break force of 520 to 868 g/cm2 and 536 to 800 g/cm2 respectively. They compared physiological and microscopical characters of microbes grown on both, agar and carrageenan containing media. The drawbacks are they have not mentioned pH of the bacteriological media and they compared physiological and microscopic characters of microbes. However, they did not carry out preparation of alkaline solid media and isolation of alkaliphiles.
M. Reeslev and A. Kjoller in their paper entitled “Comparison of biomass dry weights and radial growth rates of fungal colonies on media solidified with different gelling compounds” in Appl. Env. Microbiol. (1995) 61: 4236-4239 have described the media with different gelling agents for cultivation of three different fungi. They prepared solid media having pH 6.0 with agar, pluronic F-127, carrageenan X-4910 or carrageenan X-4910 overlaid with cellophane. They reported the similar fungal growth and visual appearance of the colonies on both gelled media, e.g., the pigmentation on carrageenan X-4910 was generally the same as that on agar. The drawbacks are preparation of media having acidic pH (6.0) using carrageenan as an alternative gelling agent to that of agar and they cultivated fungi for physiological studies. They did not prepare solid media having alkaline pH and also no mention is made on cultivation of bacteria.
Laserna et al in their paper entitled “Carrageenan from Eucheuma striatum (Schmitz) in media for fungal and yeast cultures” in Appl. Env. Microbiol. (1981), 42:174-175 have prepared plates and slants of potato dextrose for fungi and cornmeal for yeast with agar (at pH 7.1 to 7.28) and carrageenan (at pH 7.18 to 7.46) in the same concentration (1.5%) and a loopful of each inoculum was streaked on the plates and slants and incubated at 27° C. for 2 to 7 days for fungi and 30° C. for 2 days for yeast. They observed the similar growth of fungi and yeasts on agar and carrageenan containing media. The drawbacks are preparation of media having neutral pH and cultivation of fungi and yeast. They did not carry out the preparation of alkaline solid media having pH greater than 7.5 and no mention made on the cultivation of bacteria.
Rambach Alain in US patent Application No. 20030129731 dated on 10 Jul. 2003 and 20060134730 dated on 22 Jun. 2006 have disclosed a new dehydrated culture medium, soluble in cold water containing κ- or τ-carrageenan as a gelling agent. He described the method of sample analysis by mixing it with dehydrated medium, dissolving in water, and allowing to form gel followed by incubation at 37° C. for 24 to 48 h. He used the medium to identify contaminants from samples of food industries and health care hospital. The drawback of the process is that κ- or τ-carrageenans are not soluble in cold water and hence it is essential to either boil or autoclave the culture medium. Moreover, he carried out the work of detection and identification of contaminant bacteria from food industry and care hospital. Also, he did not describe alkaline solid media preparation and isolation of alkaliphiles in extreme condition.
Read Taintor in his patent (U.S. Pat. No. 7,018,828 dated 28 Mar. 2006) entitled “Microbial culture medium containing agar and τ-carrageenan”, has disclosed the culture media containing both, agar and carrageenan, for identification and antimicrobial susceptibility testing of unknown microorganisms. He used agar as a gelling agent and carrageenan as a stabilizing agent to improve the shelf life of agar containing media. The carrageenan-infused culture media resulted in a reduction of syneresis, thus providing potential improved performance of the media. The drawbacks are i) he used both, agar and carrageenan as a gelling agents, the purpose of addition of τ-carrageenan to increase the shelf life ii). He did not mention about pH of microbiological culture media. Also, he did not prepare microbiological media having high alkaline pH and no mention made on the isolation, purification and identification of alkaliphilic bacteria.
Shungu et al in their paper entitled “Gelrite as an agar substitute in bacteriological media” in Appl. Env. Microbiol. (1983) 46: 355-359 has described the preparation of blood agar (tryptic soy broth with 5% sheep blood) and selective media such as bismuth sulphite, brilliant green, eosin methylene blue and MacConkey using agar and gelrite as the gelling agents. They compared the growth of 50 different species on a variety of common as well as differential, selective and enrichment media gelled by agar and gelrite. After inoculation, the plates were incubated at 35° C. for 24 to 72 h. They reported all the test parameters like colony characteristics, biochemical reactions, haemolytic patterns, and plating efficiency were comparable on the media gelled by agar or gelrite. The drawbacks are: (1) they used gelrite as an alternative gelling agent; (2) no mention is made on pH of the media and (3) application of these media for isolation, identification and enumeration of bacteria. (4) They did not describe the use of carrageenan as a gelling agent; no mention is made on the preparation of media having alkaline pH and use of media for the isolation of alkaliphiles.
B. J. Bromke and M. Furiga in their paper entitled “Carrageenan is a desirable substitute for agar in media for growing Trichomonas vaginalis” in J Microbiol. Methods (1991) 13: 61-65 have used many commercially available gelling agents as potential substitutes for agar in media for growing a protozoan, Trichomonas vaginalis. According to them, only carrageenan (0.3 g/100 ml) fulfilled all the requirements for growth, normal morphology and proper harvesting of trichomonads with cost-efficiency. However, the drawback is that the paper deals only with the cultivation of protozoa. Also, no mention is made on suitability of κ-carrageenan as gelling agent, specifically for the isolation, purification and identification of alkaliphilic bacteria.
I. A. Abbott and F. A. Chapman in their paper entitled “Evolution of κ-carrageenan as a substitute for agar in microbiological media” in Arch. Microbiol. (1981) 128: 355-359 have mentioned that carrageenan was superior in comparison to agar with respect to transparency of the medium. They have used 71 samples of the colloid κ-carrageenan extracted from 12 seaweed species and reported that all the samples had a lower melting temperature (less than 67° C.) than agar and a gelling (setting) temperature between 16° C. and 51° C. They also added all the gelling samples did not show syneresis in the slant as well as in the plates. They observed all the test microorganisms grew well on both κ-carrageenan based media and agar containing media. The drawbacks of the paper are application of these media for cultivation of microorganisms and no mention is made about pH of the microbiological media. They did not describe the preparation of carrageenan based solid media and isolation of alkaliphilic bacteria.
P. L. Rule and A. D. Alexander in their paper entitled “Gellan gum as a substitute for agar in leptospiral media” in J. Clin. Microbiol. (1986) 23: 500-504 have described the preparation of media with gellan gum and agar as gelling agents for the cultivation of both pathogenic and saprophytic Leptospira. They observed the similar colonial growth on agar and on gellan gum media and reported that the gellan gum containing media has a long-term stability up to 9-12 months and hence preferred as a maintenance medium. The drawbacks are use of gellan gum as a gelling agent and cultivation of Leptospira. Also, they did not describe the use of carrageenan as a gelling agent, preparation of extreme alkaline media and isolation and cultivation of alkaliphiles.
Jain et al in their paper entitled “Guar gum: a cheap substitute for agar in microbial culture media” in Letts. Appl. Microbiol. (2005) 41: 345-349 have described the preparation of agar and guar gum gelled media for bacterial and fungal studies. They have cultivated 11 bacteria and 12 fungi on media solidified with either guar gum or agar. They observed the normal bacterial and fungal growth on the media gelled with guar gum and reported similar bacterial and fungal structures during the microscopical examination. They examined the bacterial enumeration studies by serial dilution and pour plate method and found that similar bacterial counts on both agar and guar gum. The drawbacks are use of guar gum as gelling agent, cultivation of bacteria and fungi and no mention is made about pH of the media. They did not describe the application of κ-carrageenan as gelling agent, preparation of alkaline solid media and isolation of alkaliphiles.
In view of the above citations, the inventors of the present invention understood the need of a solid nutrient media composition having alkaline pH, useful for isolating and identifying alkaliphilic microorganisms in pure form, which obviates the drawbacks of the mentioned citations.